Rephrasing of the title of the famous concept album “The Dark Side of the Moon” (1973) by the English progressive rock group Pink Floyd. Already used to highlight the reactivity of the heme proximal side of cytochromes towards NO (see ref.11). Here, this notion is expanded to human hemoglobin.
AHSP, α-hemoglobin stabilizing protein; Hb, hemoglobin; HbONO, NO2−-bound ferric Hb; NαHbONO, HbONO displaying nitrated 2-vinyl heme group of αHb; NHbONOα, HbONO displaying NO2−-free βHb and nitrated 2-vinyl heme group of αHb and βHb; HbONOd,p, HbONO displaying NO2−-bound to the distal and proximal heme sides of αHb and βHb, respectively; αHb, α-chains of Hb; αHbO2oxygenated αHb; αHbONO, αHb of HbONO; αHbONOd,p, αHb of HbONOd,p; βHb, β-chains of Hb; βHbONO; βHb of HbONO; βHbONOd,p, βHb of HbONOd,p; hhcytc, horse heart cytochrome c; Mb, myoglobin; sGC, soluble guanylate cyclase
For long time, ligand binding to the heme distal side has been considered as a paradigm of biochemistry, the fifth trans axial ligand of the heme–Fe atom being the proximal His residue. Ligands bind to the heme center with very different values of thermodynamic and kinetic parameters depending on the ligand chemistry, on the oxidation and coordination state of the heme-Fe atom, and on amino-acid residues building up the heme pocket. Moreover, ligand binding to heme-proteins may be modulated by homotropic and heterotropic allosteric effectors (1–8).
Recently, the possibility that NO could bind to the proximal heme side of heme-proteins came to light as the proximal HisFe axial bond can be lost upon NO binding to the distal heme coordination side (2, 9). Among others, the cleavage of the proximal HisFe axial bond occurs in the α-chains of ferrous nitrosylated human hemoglobin upon binding of heterotropic effectors switching the quaternary transition toward the T-state (2). Remarkably, the cleavage of the proximal HisFe axial bond has been reported to represent the first step of NO binding to the proximal heme coordination side (10–12).
NO binds to the proximal heme side of a periplasmic class IIa c-type cytochrome c′ from the denitrifying bacterium Alcaligenes xylosoxidans, of horse heart cytochrome c (hhcytc) complexed with cardiolipin, and of mammalian-soluble guanylate cyclase (sGC), displacing the proximal His residue and inducing the formation of a penta-coordinated heme–Fe–NO derivative (10–16). Notably, in hhcytc a transient heme–Fe–bis–NO complex precedes the formation of the penta-coordinated heme–Fe–NO species (10, 11). In mammalian sGC, data highlighting a heme–Fe–bis–NO complex are lacking and a bis–NO transition complex was postulated to indicate the fast binding of a NO molecule at the proximal heme-Fe side with the concomitant cleavage of the proximal His-Fe bond, with the subsequent discharge of the NO ligand bound to the distal heme-Fe side (12).
The hhcytc–cardiolipin complex could play either proapoptotic effects, catalyzing lipid peroxidation and the subsequent hhcytc release into the cytoplasm, or antiapoptotic actions, protecting the mitochondrion by scavenging reactive nitrogen and oxygen species and binding CO and NO that inhibit lipid peroxidation and hhcytc translocation (17).
According to the “sliding scale rule”, the multistep mechanism for NO binding to mammalian sGC substantially increases the gas ligand affinity and impairs totally O2 binding (18). The NO/O2 selectivity by mammalian sGC is crucial under physiological conditions where the NO concentration is much less than that of O2 and NO dioxygenation is unwanted (19).
Remarkably, nitrosylation of the proximal heme–Fe side of heme-proteins has been postulated to be relevant in several functions including: i) discrimination between ligands (e.g., NO, CO, and O2), ii) initiation of specific gas-dependent signaling pathways, and iii) selective scavenging of reactive nitrogen and oxygen species (10–19).
Interestingly, a ligand-binding pocket has been created on the heme proximal side in quadruple mutants of porcine myoglobin (Mb) by site-directed mutagenesis, the proximal HisF8 residue having been replaced by Phe. The affinity of CO and cyanide for the heme proximal coordination side of porcine Mb quadruple mutants is similar to that for the heme distal pocket of wild-type Mb; however, the polar nature of the heme proximal pocket is at the root of the rapid oxidation of the heme–Fe atom preventing reversible O2 binding (20).
Here, the structural bases for O2 and NO2− binding to the proximal heme side of α- and β-chains of ferrous and ferric human hemoglobin (Hb), respectively, are reviewed. This suggests that: i) Hb may utilize both heme distal and proximal sides for ligand discrimination, ii) draws attention to the nonequivalence of the α- and β-subunits, and iii) highlights the possibility that partially unfolded Hb derivatives may display transient reactivity properties different from those of the native protein.
O2 Binding TO α-Chains OF Partially Unfolded Ferrous Human Hb
Hb, the O2 carrying and delivery system in humans, is an allosterically modulated hetero-tetramer formed by two α- and two β-chains (αHb and βHb, respectively) (2). Free αHb is an unstable monomer prone to heme–Fe oxidation and precipitation, likely contributing to the pathophysiology of several blood disorders including β-thalassemia. On the other hand, βHb forms a relatively stable homotetramer (21, 22).
The molecular chaperone α-hemoglobin-stabilizing protein (AHSP) is an erythroid protein that binds the α-globin polypeptide and several forms of αHb, in the absence of βHb, to maintain the αHb structure, to avoid αHb precipitation, and to limit αHb pro-oxidant activity both in vitro and in vivo. When available, βHb binds more avidly to αHb than AHSP, displacing AHSP and forming the Hb tetramer (23–29).
AHSP and oxygenated αHb (αHbO2) form a 1:1 binary complex (23). Both proteins are exclusively in the α-helical conformation, AHSP adopting an elongated three-helix bundle and αHb comprising six α-helices. The interface of the AHSP–αHbO2 complex primarily involves four α-helices, two from each protein. AHSP contacts G and H α-helices of αHbO2 (25, 26). Remarkably, the G and H α-helices of αHb are the primary structural elements that interact with βHb to form the α1–β1 Hb complex. Superimposition of the AHSP–αHbO2 complex onto the α1–β1 Hb dimer reveals that segments of the α1 and α2 helices of AHSP superimpose with G and H α-helices of βHb, forming similar interactions with αHb (26). This partially explains the reasons why binding of αHb by βHb dissociates AHSP from the AHSP–αHb complex (26).
AHSP binds to αHbO2 on the opposite side of the heme pocket, inducing dramatic conformational changes in αHb, with the entire F helix becoming flexible and disordered. Compared to oxygenated Hb (30), the heme–Fe atom and the heme group of the AHSP–αHbO2 complex slides over a distance of approximately 3.1 Å. Accordingly, the amino acid residues that coordinate the heme–Fe group in the AHSP–αHbO2 complex undergo significant rearrangements. In particular, the sixth coordination position of the heme–Fe atom is positioned facing the F α-helix of αHbO2. Strikingly, although the heme–Fe group of αHb is invariably bound by the proximal His(87)F8 residue in most structures of Hb (31) (Fig. 1, panel αHbO2), the distal His(58)E7 residue, rather than the proximal His(87)F8 side chain, coordinates the heme–Fe atom, with a distance of 2.13 Å between the Nε atom of the His(58)E7 residue and the ferrous heme–Fe atom. Moreover, the O2 molecule, rather than the proximal His(87)F8 residue, represents the trans axial ligand, the distance between the O1 atom of O2 and the heme–Fe atom being 2.74 Å (25) (Fig. 1, panel AHSP–αHbO2). In other words, O2 binds to the proximal heme–Fe side of αHb complexed with AHSP, the distal His(58)E7 residue being coordinated to the heme–Fe atom and representing the “proximal heme residue” (25, 31).
In the AHSP–αHbO2 complex, the O2 molecule is solvent exposed, resulting in a fast heme–Fe oxidation rate. In the resulting AHSP-bound ferric αHb, the proximal His(87)F8 residue of αHb serves as the trans ligand for the sixth coordination position of the heme–Fe atom, the distal His(58)E7 residue representing the fifth coordination ligand (26). Thus, AHSP binding to αHbO2 facilitates the oxidation of the heme–Fe atom and sequesters the oxidized heme in the hexa-coordinated state. This inhibits heme loss from αHb and redox chemistry catalysis, thus preventing cell damage (25, 26).
Nonequivalent NO2− Recognition by α- and β-Chains of Ferric Human Hb
Ferrous human Hb is pivotal for O2 transport; in contrast, the ferric derivative does not bind O2. Moreover, in Hb valency hybrids ferric heme(s) shifts the R-to-T allosteric transition toward the high-affinity state impairing O2 release (2, 8). Therefore, factors that facilitate Hb oxidation, such as NO2− are relevant from the health viewpoint (21, 32). Remarkably, NO2− is recommended in the therapeutic treatment of cyanide poisoning in combination with amyl nitrite and thiosulfate. NO2− antidotal properties were initially attributed to the induction of ferric Hb, removing cyanide from cytochrome c oxidase, and later to a NO-mediated hemodynamic effect (33). Notably, although the NO2− concentration in plasma ranges between 1.3 and 13 μM (34), during the therapeutic treatment of cyanide poisoning the NO2− concentration can reach millimolar plasma concentrations and 10 mM levels at the site of the intravenous administration. However, as NO2− in whole blood is very rapidly oxidized to NO3− (>95% in 1 h), the therapeutic treatment must be repeated if symptoms of cyanide poisoning recur (33).
The complexity of the Hb–NO2− interaction depends on the capability of NO2− i) to be reduced to NO, ii) to oxidize the ferrous heme–Fe atom, iii) to bind to the ferric heme–Fe atom, iv) to participate to the formation of N2O3, v) to nitrate the ferric heme, and vi) to denaturate Hb with the concomitant release of the ferric heme (35–42).
Although the crystallization conditions (e.g., the NO2− concentration was 0.16 M, and the soaking time of Hb crystals with NO2− ranged between 10 min and 1 week) (42, 43) are very different from those of pathophysiological situations (33, 34), four reaction products following NO2− binding to ferric and ferrous Hb have recently been characterized by X-ray crystallography, highlighting the unusual heme–Fe–NO2− binding geometries (42, 43).
In crystals of NO2−-bound ferric Hb (HbONO) (obtained by soaking ferric Hb crystals at pH 6.8 with NO2− for 10 min at room temperature), NO2− adopts the uncommon O-nitrito binding mode in both αHb and βHb. A near-identical structure of HbONO with O-nitrito ligands was observed in crystals obtained from the ferric Hb–NO2− solution. In αHb of HbONO (αHbONO), the Fe–O distance and the Fe–N(His(87)F8) distance are both 2.0 Å. The NO2− O1 atom is within hydrogen bonding distance (2.9 Å) of the Nε atom of the distal His(58)E7 residue (Fig. 2, panel αHbONO). In βHb of HbONO (βHbONO), the Fe–O distance is 1.9 Å and the Fe–N(His(92)F8) distance is 2.0 Å. The nitrite O1 and N atoms are within hydrogen bonding distance (2.9 and 3.2 Å, respectively) of the Nε atom of the distal His(63)E7 residue (Fig. 2, panel βHbONO). However, NO2− conformations in αHbONO and βHbONO are different, reflecting subtle effects of the distal HisE7 in orienting the NO2− ligand. In αHbONO, the Fe–O–N–O moiety is trans with a torsion angle of 174°, the O–N–O angle being 110°. On the other hand, in βHbONO, the Fe–O–N–O complex is in a distorted cis-like conformation with a torsion angle of −91° and an O–N–O angle of 113°. Moreover, the terminal nitrite atom is directed away from the distal His(63)E7 residue of βHbONO, making the shortest contact with the Cγ atom of distal Val(67)E11 (3.2 Å) (43).
The reaction of ferric Hb crystals with NO2− at pH 6.8 for 16 h results in the formation of a red-green product, named NαHbONO. The NO2− binding mode to the heme distal pocket of both αHb and βHb is similar to that observed for HbONO. At variance with HbONO, the long-time incubation of ferric Hb crystals with NO2− leads to the nitration of the 2-vinyl heme group in αHb only. The covalent modification of the vinyl heme–Fe substituent with NO2− is regiospecific, the 2-nitrovinyl moiety being essentially coplanar with the adjacent pyrrole ring, suggesting extended conjugation with this group (42).
The reaction of ferric Hb crystals with NO2− at pH 6.5 for 1 week results in the formation of a green crystalline product, named NHbONOα. The bulk features of the heme-binding pocket of αHb of NHbONOα are similar to those reported for NαHbONO. However, the heme–Fe group of βHb is also nitrated at the 2-vinyl position. Moreover, the heme–Fe atom of βHb is penta-coordinated, as no electron density for the NO2− ligand has been observed (42).
Crystallization of deoxygenated Hb in the presence of excess NO2− at pH 7.0 gave, after 3 days, a product, named HbONOd,p, showing unprecedented features in Hb structural biology. The NO2− binding mode to αHb of HbONOd,p (αHbONOd,p) (Fig. 2, panel αHbONOd,p) is closely similar to that observed in HbONO, NαHbONO, and NHbONOα. In fact, NO2− binds to αHb in the transO-binding mode and the O1 atom is hydrogen bonded to the Nε atom of the distal His(58)E7 residue. Moreover, the 2-vinyl heme group in αHb is not nitrated (42). In contrast, the βHb of HbONOd,p (βHbONOd,p) exhibit major differences from Hb structures reported in the Protein Data Bank (31). In fact, βHbONOd,p exhibits: i) a large lateral heme sliding (∼4.8 Å) toward the protein exterior; ii) heme stabilization by the formation of the distal His(63)E7Fe bond (2.3 Å); iii) loss of the proximal His(92)F8Fe bond (the closest nonbonding N(His(92)F8) C(shifted heme pyrrole) distance being 2.8 Å); and iv) most unexpectedly, the replacement of the proximal His(92)F8Fe bond by the FeNO2− bond. The Fe–O (NO2−) distance is 3.0 Å (Fig. 2, panel βHbONOd,p). In other words, in the βHbONOd,p, the “inversion” of the proximal-to-distal His heme side occurs. In fact, the exogenous ligand (i.e., NO2−) is bound to the heme–Fe atom at the classically called heme proximal position instead of His(92)F8, and the so-called distal His(63)E7 corresponds to the proximal heme–Fe atom axial ligand (42).
As a whole, the multiple binding modes of NO2− to and αHb and βHb highlight the nonequivalent functional properties of Hb chains at least in the ferric form (42, 43). Notably, nitrite binding to the Hb tetramer displays biphasic kinetics similar to those observed for ligation of isolated αHb and βHb (35). Moreover, the HbONOd,p species, in which the NO2−-bound heme–Fe group of βHb is displaced ∼4.8 Å from its original position, may represent an intermediate of Hb unfolding. In fact, the heme displacement would lead to eventual heme removal and protein destabilization (42).
Hb, playing a pivotal role in life, is a classic paradigm for the study of protein structure–function relationships (1, 2, 8, 21). The unusual O2 and NO2− binding mode in AHSP–αHbO2 and βHbONOd,p, respectively, highlights new modulation mechanisms contributing significantly to the understanding of Hb homeostatic regulation in living organisms (25, 42).
Here are some lessons learned in looking at Hb reactivity. Hb may utilize both heme distal and proximal sides for ligand discrimination, as observed for binding of the exogenous O2 and the endogenous His(87)F8 ligand. In fact, O2 binding to the heme proximal side of ferrous αHb of AHSP–αHbO2 leads to the heme–Fe atom oxidation followed by the formation of the inert ferric bis-histidyl adduct (25). Moreover, NO2− binding to the heme proximal side of ferric βHbONOd,p represents a unique case (42); in fact, anionic ligands most invariably bind to the heme distal side of ferric Hb (31). Furthermore, NO2− binding to ferric Hb highlights the ligand-binding nonequivalence of αHb and βHb. In fact, in NHbONOα NO2− binds only to αHb, and in HbONOd,p NO2− binds to heme-distal and heme-proximal side of αHb and βHb, respectively (42). The different binding mode of NO2− to αHb and βHb in HbONOd,p reflects the sliding movement of the heme toward the βHb exterior, representing an intermediate of Hb unfolding (42). Ligand-dependent heme sliding may be a general mechanism for ligand-binding modulation as also observed for carbonylation of murine neuroglobin (44).
Finally, the possibility that partially unfolded Hb derivatives may display transient ligand-binding properties different from those of the native globin and of the unfolded protein may represent a case of “chronosteric effects” (45–47). Remarkably, the Hb reactivity toward CO increases transiently at low pH (<4), preceding the irreversible protein denaturation. This reflects the protonation of the Nε atom of the HisF8 residue and the cleavage of the proximal axial heme–Fe–Nε bond, representing the first step of protein unfolding. In turn, the low reactive penta-coordinated heme becomes tetra-coordinated and highly reactive, the heme–Fe atom shifting from the “out” to the “in” plane geometry. This highlights the crucial role of the interaction(s) of the heme on the proximal side in accounting for the difference in the ligand reactivity between the two quaternary R and T conformations of Hb (48).
Dr. Loris Leboffe is supported by a grant from the National Institute of Biostructures and Biosystems (INBB) of Italy.